Paroxysmal Nocturnal Hemoglobinuria (PNH) is an acquired hematopoietic stem cell disease (1, 2) that is caused by a somatic mutation of the X-linked phosphatidyl-inositol glycan (PIG-A) gene (3, 4). Since the PIG-A protein is involved in the initial stage of synthesis of the glycosylphosphatidyl-inositol (GPI) anchor (5, 6), these defects result in partial or absolute deficiency of all GPI-linked proteins/glycoproteins in a clone of hematopoietic stem cells (7–9). Expansion of PNH-like clones can also be detected in cases of aplastic/hypoplastic/myelodysplastic syndromes caused by an immune-mediated failure of normal hematopoiesis or defect in hematopoietic stem cells (10).
Typical clinical features of PNH are: bone marrow failure of variable severity, thrombosis in unusual sites, chronic intravascular hemolytic anemia that leads to hemoglobinuria, iron deficiency anemia, and increased incidence of acute myeloid leukemia (11). Activated serum complement was shown to play an important role in the hemolytic anemia and two GPI-linked structures CD55 (Decay Accelerating Factor, DAF) and CD59 (Membrane Inhibitor of reactive lysis, MIRL) on red blood cells play an important role in the control of complement (12, 13). While the absence or partial expression of CD55 and CD59 is specific for PNH, CD59 deficiency alone appears to be responsible for hemolysis and other clinical symptoms of PNH (14, 15).
Diagnosis and follow-up of PNH patients has been greatly improved by the advent of flow cytometry-based assays (16–18, reviewed in Refs.19 and20). Because of the historical role of red blood cells in the diagnosis of PNH (21), most flow cytometric approaches have involved the staining of patients' red blood cells with CD55 and CD59 (18, 22). Flow cytometric analysis of RBCs in untransfused patients can be used to quantitate the Type III (complete deficiency), type II (partial deficiency), and type I (normal expression) clones (19, 20). There are several potential problems with this approach; the lysis of Type III RBCs and or the presence of recently transfused normal RBCs significantly reduces the ability to detect PNH RBCs. As PNH is a rare disease, deficiency of at least two GPI-linked structures on more than one lineage, typically, neutrophils, is an additionally required criterion to establish a diagnosis. In situations of prior hemolysis and/or recent red cell transfusion, detection of PNH granulocytes best reflects the size of the PNH clone. However, severe neutropenia in some patients makes it difficult to identify the target population using light scatter characteristics alone. Furthermore, in a small number of PNH patients, only neutrophils are affected (18). Because of their prolonged life span, lymphocytes are not normally a good target population for analysis because only those arising post-disease onset will be deficient in GPI-linked structures; the majority will express GPI proteins whereas neutrophils, monocytes, and red cells do not.
Although a rare disease, the PNH assay is frequently requested and in the 2004 calendar year, we screened 141 samples for PNH and detected only three bona fide cases of PNH. At our institutions, we used the two-part Biocytex™ kit (Biocytex, Marseille, France), a quantitative immunocytometric approach that utilizes fluorescent beads to set windows of analysis and the staining of CD55 and CD59 on both red cells (Redquant™) and neutrophils (Cellquant™). The Biocytex assay is labor-intensive to set up, given the low frequency of the disease, and the Cellquant assay for neutrophils must be set up within 8 h of sample draw. Given the large number of referral samples that are analyzed at our institution, we were unable to meet the latter criterion on a number of samples.
An alternative approach that makes it possible to perform less extensive testing for the diagnosis of PNH has more recently been suggested that utilizes the bacterial toxin Aerolysin. Proaerolysin is a 52-kDa protein secreted by Aeromonas hydrophila. After proteolytic nicking at the C-terminus, the active form, Aerolysin is generated that binds to cell surface structures and oligomerizes, forming channels that result in cell lysis (23). Aerolysin does not lyse PNH cells and it was shown that the toxin bound to the GPI moiety of GPI-linked structures (24, 25). Initially, this reagent was used to enrich rare GPI-negative PNH clones. Subsequently, a fluorochrome-conjugated (Alexa 488) version of a non-lysing, mutated form of proaerolysin (FLAER) was generated that retained specificity for GPI-linked structures without causing cell lysis. FLAER is more sensitive than CD59 at detecting small abnormal granulocyte populations in patients to a level of approximately 0.5%. It was claimed that FLAER gave a more accurate assessment of the GPI anchor deficit in PNH (26). However, FLAER cannot be used to assess PNH clones in the erythrocyte lineage, since the latter do not possess surface-bound proteolytic enzymes needed to process the Proaerolysin. Further excluding the utility of aerolysin in the detection of GPI-linked structures on red cells is the observation that glycophorin binds weakly to proaerolysin despite the fact that this major red cell glycoprotein is not GPI-linked (27).
While simple flow-based FLAER assays for PNH have been developed (26, 28), the ability to detect rare PNH clones in closely related diseases such as aplastic anemia and myelodysplasia would be enhanced by a more sophisticated multi-parameter approach. In this study, we developed such an approach based upon the staining of a single pre-lysed blood sample with antibodies to CD14 (GPI-linked), CD33 (non-GPI-linked), CD45, and FLAER. In comparison with our predicate method, the assay is more sensitive, less expensive, and takes only 40 min from sample draw. Interestingly, we have also identified aberrant FLAER expression in several cases of undiagnosed acute myeloid leukemia, sent initially for PNH testing.
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- LITERATURE CITED
Paroxysmal Nocturnal Hemoglobinuria is a rare acquired disorder of the hematopoietic stem cell (1–9). Despite its rarity, this test was performed in the UHN Clinical Flow Cytometry laboratory 141 and 164 times in the 2004 and 2005 calendar years respectively. Only three samples were unequivocally diagnosed as PNH using the Biocytex Cellquant™ and Redquant™ kits in 2004. Performance of this assay is labor intensive and costly requiring an average of more than 2 h to set up and analyze. Furthermore, the Cellquant™ assay for neutrophils needs to be performed within 8 h of sample draw and as a large reference centre, not all our potential PNH samples could be tested within this time frame. It is not known if any PNH-positive samples were undiagnosed while this methodology was in use because of “age-of-sample” issues. In such cases, the Redquant™ assay only was performed, but the transfusion status of the patient was usually unknown at the time of testing. In cases where the Redquant™ assay generated data suggestive of PNH, a repeat sample was required to perform both parts of the kit for accurate diagnosis.
In this paper, we describe a rapid and technically simple assay utilizing FLAER in combination with CD33, CD45, and the GPI-linked structure CD14 that obviates many of the issues noted above. This assay allows the simultaneous detection of PNH clones in monocyte and granulocyte lineages. The assay takes less than 1 h from sample receipt and can be performed on a variety of bench-top instruments with four color detectors. Special software is not required for acquisition or data analysis and overall, the method is inexpensive compared to the commercial method. The assay is robust in that expression of the PNH phenotype on monocytes (FLAER-negative, CD14-negative, CD33-bright) and granulocytes (FLAER-negative, CD14-negative, CD33-weak) is stable at least up to 24 h and perhaps up to 48 h post sample draw. Similarly, non-PNH samples remain stable to analysis with this combination for 24 h and beyond and we have not detected PNH clones in any normal samples analyzed up to 48 h post sample draw. Since development and deployment of the FLAER assay in October 2004, 206 samples were tested up to December 2005 and 5 previously undiagnosed PNH cases were detected.
The assay appears to be more sensitive than the method used hitherto in our laboratory. Other previously described assays that depended on single parameter FLAER staining have claimed an assay sensitivity of about 1% (26, 28). We expect that the multiparameter assay described herein will be at least as sensitive although we have not been able to formally test this on appropriate patient samples. In a number of referred samples with known PNH or other hematological diseases, we identified abnormal FLAER expression in minor populations as low as 4%.
One previously diagnosed PNH patient (PL) came in for further monitoring on two occasions and found to still have PNH monocyte and granulocyte populations in the 4–5% range. Additionally, we have been able to follow a patient (AL) with aplastic anemia over a 14-month period and were able to monitor PNH monocyte and neutrophil clones down to the 1.2–1.5% levels respectively in the most recent sample. We also identified two more patients previously diagnosed as aplastic anemia (AL and JD) with low numbers of PNH monocyte and granulocyte clones.
As expected, monocyte and granulocyte lineages showed similar proportions of PNH versus normal clones in all but one PNH sample analyzed to date. In the latter case (AS), a difference of about 20% was noted between the more numerous monocyte population and the granulocytes. The explanation for this is unknown. In all cases in which the red cells were analyzed with the simple CD59 staining method, the proportion of PNH (type II and/or Type III) versus normal clones was always very much lower than that detected in the monocyte and/or granulocyte lineages. Potential explanations for this general observation include hemolysis of PNH red cells, particularly those with a Type III phenotype, or a history of recent red blood cell transfusion. However, in at least one case (VT) the proportion of PNH red cells (type II plus type III) was always <50% of the PNH clones detectable in either monocyte and/or granulocyte lineages even though this patient had never received a red blood cell transfusion. Thus, even in this non-transfused patient, enumeration of white blood cell lineages (granulocytes and/or monocytes) with the FLAER assay more reliably quantifies the size of the PNH clone at the hematopoietic stem cell level than the evaluation of CD55 and/or CD59 staining of red blood cells. Regardless, the CD59 assay on RBCs still has clinical utility in the monitoring of increased PNH RBCs in the context of newer therapies such as Eculizumab, a humanized monoclonal antibody that blocks the hemolysis of PNH RBCs (29).
The multiparameter FLAER-based assay described herein has also identified abnormally reduced FLAER expression in leukemic blast populations in several patient samples submitted for “PNH testing”. In each case, the granulocyte population exhibited a normal staining pattern with FLAER, CD14, and CD33, quickly ruling out a diagnosis of PNH. In contrast, the CD33-bright fraction exhibited anomalies with regard to one or more of the expression of GPI-linked CD14 and/or CD45 and light scatter characteristics in addition to the unusual expression of FLAER. Reduced FLAER expression in these cases likely represents a marker of immaturity with reduced expression by leukemic blasts of GPI-linked proteins. As patients with either PNH or acute leukemia may present as pancytopenia, the flow cytometrist needs to be aware that the presence of blasts in a sample may lead to erroneous diagnosis of PNH based on FLAER expression as the sole marker for PNH detection, further supporting the requirement for a multiparameter approach. It is also recommended that whenever non-PNH hematologic abnormalities are detected with the FLAER assay, a smear of the blood sample should be made to confirm the presence of abnormal blast populations.
One important issue that needs to be addressed when developing assays for routine deployment in clinical flow laboratories is the availability of suitable material for Proficiency Testing and Quality Assurance purposes. In most jurisdictions, clinical laboratories are subjected to Laboratory Licensing through Quality Assurance and Proficiency Testing schemes. Finding standardized PNH sample material in sufficient quantities that is stable and amenable for shipping to the large number of clinical laboratories performing testing for PNH is highly problematic given the rarity of this disease. Recently however, stabilized blood samples from bona fide PNH patients have been made available by the UK NEQAS (National External Quality Assurance Scheme) organization that in preliminary testing, has been shown to be suitable for both the FLAER and CD59 red cell assays described here (unpublished data). Although preliminary, these observations suggest that if such material can be made available in sufficient quantities, a major hurdle in the standardization of PNH testing can be overcome.
In summary, we have combined FLAER with multiparameter flow cytometry to develop an improved assay for diagnosis and monitoring of PNH and detection of PNH clones in a variety of hematological diseases. This assay is sensitive, stable, obviates many of the drawbacks of other flow-based assays and represents a very simple and efficient means to detect PNH in the clinical flow laboratory.